CA2595960A1 - Antilymphocyte antibody induction by combination of an s1p receptor agonist/modulator and of immunosuppressive drugs - Google Patents

Abstract

An immunosuppressive treatment combining a S1 P receptor modulator, one or more immunosuppressive drug(s) and an antilymphocyte antibody in the course of the treatment of a transplant recipient prolongs the survival of a transplant allograft.

Description

Antilymphocyte Antibody Induction The present invention relates to an immunosuppressive treatment combining an immunosuppressive drug, an S1 P receptor modulator and an antilymphocyte antibody, particularly in the course of the treatment of a transplant patient.

Current maintenance immunosuppressive therapy, for example after kidney transplantation, combines calcineurin inhibitors (cyclosporine or tacrolimus) with one or more immunosuppressive drugs, including corticosteroids, azathioprine (AZA), mycophenolate mofetil, mycophenolate sodium, or macrolide immunosuppressants (everolimus, sirolimus).
These combinations have been developed in an effort to optimize the prevention of acute rejection episodes, and to minimize or avoid adverse effects. These efforts have yielded significant progress, but there is still a need for improvement with regard to rejection rates and side effects, in particular.

It has now surprisingly been found that a combination of an S1 P receptor agonist with one or more immunosuppressive drugs including antilymphocyte antibody treatment will provide further unexpected benefits. In particular, the rejection episodes, e.g. after renal or heart transplantation, are reduced while keeping the dosage of the other agents at a minimum level, thereby resulting in improved tolerability.

Accordingly it is provided a method of inhibiting allograft rejection in a recipient, said method comprising administering to said recipient simultaneously or sequentially a therapeutically effective amount of a S1 P receptor modulator in combination with one or more immunosuppressive drugs and antilymphocyte antibody.

Preferably the method according to the invention may be used for treating renal transplants.
S1 P receptor modulators or agonists are compounds which target one or more sphingosine 1-phosphate receptors, e.g. S1 P1 to S1 P5. The term modulation is meant to cover agonism or functional antagonism of the S1 P receptor(s). Modulator or agonist binding to a S1 P
receptor may e.g. result in activation, internalization or desensitization of the receptor(s).
This may be associated with a modulation of S1 P receptor(s) signaling via G
proteins, association or dissociation of different G proteins, changes in the interaction of G proteins with the S1 P receptor(s), altered regulation of the G proteins by RGS
(regulators of G
protein signaling) proteins, increased phosphorylation of the modulator-occupied receptor, and/or activation of downstream signaling pathways/kinases.

The binding affinity of S1 P receptor agonists or modulators to individual human S1 P
receptors may be determined in following assay:
S1 P receptor agonist or modulator activities of compounds are tested on the human SIP
receptors S1 P,, S1 P2, S1 P3, S1 P4 and S1 P5. Functional receptor activation is assessed by quantifying compound induced GTP [y-35S] binding to membrane protein prepared from transfected CHO or RH7777 cells stably expressing the appropriate human S1 P
receptor.
The assay technology used is SPA (scintillation proximity based assay).
Briefly, DMSO
dissolved compounds are serially diluted and added to SPA- bead (Amersham-Pharmacia) immobilised S1 P receptor expressing membrane protein (10-20 g/well) in the presence of 50 mM Hepes, 100 mM NaCI, 10 mM MgC12, 10 pM GDP, 0.1 % fat free BSA and 0.2 nM
GTP [7-35S] (1200 Ci/mmol). After incubation in 96 well microtiterplates at RT
for.120 min, unbound GTP [7-35S] is separated by a centrifugation step. Luminescence of SPA
beads triggered by membrane bound GTP [7-35S] is quantified with a TOPcount plate reader (Packard). EC50s are calculated using standard curve fitting software. In this assay, the S1 P
receptor modulators or agonists preferably have a binding affinity to SIP
receptor <50 nM.
Preferred S1 P receptor agonists or modulators are e.g. compounds which in addition to their S1 P binding properties also have accelerating lymphocyte homing properties, e.g.
compounds which elicit a lymphopenia resulting from a re-distribution, preferably reversible, of lymphocytes from circulation to secondary lymphatic tissue, without evoking a generalized immunosuppression. Naive cells are sequestered; CD4 and CD8 T-cells and B-cells from the blood are stimulated to migrate into lymph nodes (LN) and Peyer's patches (PP).

The lymphocyte homing property may be measured in following Blood Lymphocyte Depletion assay:
A S1 P receptor agonist or modulator or the vehicle is administered orally by gavage to rats.
Tail blood for hematological monitoring is obtained on day -1 to give the baseline individual values, and at 2, 6, 24, 48 and 72 hours after application. In this assay, the S1 P receptor agonist or modulator depletes peripheral blood lymphocytes, e.g. by 50%, when administered at a dose of e.g. < 20 mg/kg.

Examples of appropriate S1 P receptor modulators or agonists are, for example:
- Compounds as disclosed in EP627406A1, e.g. a compound of formula I

H~OR3 I

Ri wherein R, is a straight- or branched (C12_22)chain - which may have in the chain a bond or a hetero atom selected from a double bond, a triple bond, 0, S, NR6, wherein R6 is H, C1_4alkyl, aryl-Cl-4alkyl, acyl or (C1_4alkoxy)carbonyl, and carbonyl, and/or - which may have as a substituent CI-4alkoxy, C2_4alkenyloxy, C2-,alkynyloxy, aryIC1_4alkyl-oxy, acyl, CI-4alkylamino, C1_4alkylthio, acylamino, (CI_4alkoxy)carbonyl, (CI_4alkoxy)-carbonylamino, acyloxy, (CI_4alkyl)carbamoyl, nitro, halogen, amino, hydroxyimino, hydroxy or carboxy; or R, is - a phenylalkyl wherein alkyl is a straight- or branched (C6_20)carbon chain;
or - a phenylalkyl wherein alkyl is a straight- or branched (C1_30)carbon chain wherein said phenylalkyl is substituted by - a straight- or branched (C6_20)carbon chain optionally substituted by halogen, - a straight- or branched (C6_20)alkoxy chain optionally substitued by halogen, - a straight- or branched (C6_2o)alkenyloxy, - phenyl-Cl_14alkoxy, halophenyl-CI.4alkoxy, phenyl-C1_14alkoxy-C,_14alkyl, phenoxy-C1_4alkoxy or phenoxy-C1_4alkyl, - cycloalkylalkyl substituted by C6_20alkyl, - heteroarylalkyl substituted by C6_20alkyl, - ----- - - - -Tie erf ocyc i(6_~o ky or - heterocyclic alkyl substituted by C2_20alkyl, and wherein the alkyl moiety may have - in the carbon chain, a bond or a heteroatom selected from a double bond, a triple bond, 0, S, sulfinyl, sulfonyl, or NR6, wherein R6 is as defined above, and - as a substituent C1_4alkoxy, C2_4alkenyloxy, C2_4alkynyloxy, aryIC1_4alkyloxy, acyl, C1_4alkyl-amino, C,-4alkylthio, acylamino, (CI_4alkoxy)carbonyl, (C1_4alkoxy)carbonylamino, acyloxy, (Cl_4alkyl)carbamoyl; nitro, halogen, amino, hydroxy or carboxy, and each of R2, R3, R4 and R5, independently, is H, C1_4 alkyl or acyl or a pharmaceutically acceptable salt or hydrate thereof;

- Compounds as disclosed in EP 1002792A1, e.g. a compound of formula II

R'4R'5N-IC-(CH2)2~ ~ C (CH2)m ~ ~ II
CHzOR'2 wherein m is 1 to 9 and each of R'2, R'3, R'4 and R'5, independently, is H, C1_6alkyl or acyl, or a pharmaceutically acceptable salt or hydrate thereof;
- Compounds as disclosed in EP0778263 Al, e.g. a compound of formula III
N R" R"
~ 1 2 -~ Y
W-C -Z z ~ ' / ' I I I
I
(CH2)m.OR%
wherein W is H; C1_6alkyl, C2_6alkenyl or C2_6alkynyl; unsubstituted or by OH
substituted phenyl; R"4O(CH2)n; or C1_6alkyl substituted by 1 to 3 substituents selected from the group consisting of halogen, C3_$cycloalkyl, phenyl and phenyl substituted by OH;
X is H or unsubstituted or substituted straight chain alkyl having a number p of carbon atoms or unsubstituted or substituted straight chain alkoxy having a number (p-1) of carbon atoms, e.g. substituted by 1 to 3 substitutents selected from the group consisting of CJ_6alkyl, OH, C,_salkoxy, acyloxy, amino, Cl_6alkylamino, acylamino, oxo, haloC1_6alkyl, halogen, unsubstituted phenyl and phenyl substituted by 1 to 3 substituents selected from the group consisting of C1_6alkyl, OH, C1_6alkoxy, acyl, acyloxy, amino, Cl_6alkylamino, acylamino, haloC1_6alkyl and halogen; Y is H, C1_6alkyl, OH, C1_6alkoxy, acyl, acyloxy, amino, Cl_ 6alkylamino, acylamino, haloC1_6alkyl or halogen, Z2 is a single bond or a straight chain alkylene having a number or carbon atoms of q, each of p and q, independently, is an integer of 1 to 20, with the proviso of 6<p+q<23, m' is 1, 2 or 3, n is 2 or 3, each of R",, R"2, R"3 and R"4, independently, is H, C1_4alkyl or acyl, or a pharmaceutically acceptable salt or hydrate thereof, - Compbunds as disclosed in WO02/18395, e.g. a compound of formula lVa or lVb -5- ?H2R3b i 1a i H2R3a R1a I (R2a)2N-C-CH2_)(_a_ P =0 (R2a)2N C-CH21~- P =0 I ~

CH2 R1b CH2 R1b CH2 or CH2 \ I ~

(CH2)7CH3 IVa Ya R4a lVb wherein Xa is 0, S, NR1s or a group -(CH2)õa , which group is unsubstituted or substituted by 1 to 4 halogen; na is 1 or 2, R1s is H or (C1_4)alkyl, which alkyl is unsubstituted or substituted by halogen; R1a is H, OH, (C1_4)alkyl or O(C1_,)alkyl wherein alkyl is unsubstituted or substituted by 1 to 3 halogen; R1b is H, OH or (C1.4)alkyl, wherein alkyl is unsubstituted or substituted by halogen; each R2a is independently selected from H or (C1_4)alkyl, which alkyl is unsubstituted or substitued by halogen; R3a is H, OH, halogen or O(C1_4)alkyl wherein alkyl is unsubstituted or substituted by halogen; and R3b is H, OH, halogen, (C1_4)alkyl wherein alkyl is unsubstituted or substituted by hydroxy, or O(C1-4)alkyi wherein alkyl is unsubstituted or substituted by halogen; Ya is -CH2-, -C(O)-, -CH(OH)-, -C(=NOH)-, 0 or S, and R4a is (C4_14)alkyl or (C4_14)alkenyl;
or a pharmaceutically acceptable salt or hydrate thereof;
- Compounds as disclosed in WO02/06268AI, e.g. a compound of formula VII
NRidR2d R6d R7d R4d (CH2)na~\ Xd Yd Rsd VII
R3dO

wherein each of Rid and R2d, independently, is H or an amino-protecting group;
R3d is hydrogen, a hydroxy-protecting group or a residue of formula _ P < ORsd II OR8d R4d is C1_4alkyl;
nd is an integer of 1 to 6;

Xd is ethylene, vinylene, ethynylene, a group having a formula - D-CH2-(wherein D is carbonyl, - CH(OH)-, 0, S or N), aryl or aryl substituted by up to three substitutents selected from group a as defined hereinafter;
Yd is single bond, Cl_loalkylene, Cl-loalkylene which is substituted by up to three substitutents selected from groups a and b, C,_,oalkylene having 0 or S in the middle or end of the carbon chain, or Cl-loalkylene having 0 or S in the middle or end of the carbon chain which is substituted by up to three substituents selected from groups a and b;
R5d is hydrogen, C3_6cycloalkyl, aryl, heterocyclic group, C3_6cycloalkyl substituted by up to three substituents selected from groups a and b, aryl substituted by up to three substituents selected from groups a and b, or heterocyclic group substituted by up to three substituents selected from groups a and b;
each of R6d and R7d, independently, is H or a substituent selected from group a;
each of R8d and R9d, independently, is H or C14alkyl optionally substituted by halogen;
<group a > is halogen, lower alkyl, halogeno lower alkyl, lower alkoxy, lower alkylthio, carboxyl, lower alkoxycarbonyl, hydroxy, lower aliphatic acyl, amino, mono-lower alkylamino, di-C1_4alkylamino, acylamino, cyano or nitro; and <group b> is C3_6cycloalkyl, aryl or heterocyclic group, each being optionally substituted by up to three substituents selected from group a;
with the proviso that when R5d is hydrogen, Yd is a either a single bond or linear Cl_lo alkylene, or a pharmacologically acceptable salt, ester or hydrate thereof;

-Compounds as disclosed in JP-14316985 (JP2002316985), e.g. a compound of formula VIII
N R R Rse Xe ~'e Rse 1e 2e R4e (CH2)ee VIII
R3eO F''7e wherein R1e,R2e,R3e,R4e,R5e,R6e,R,ei ne, Xe and Ye are as disclosed in JP-14316985;
or a pharmacologically acceptable salt, ester or hydrate thereof;
-Compounds as disclosed in WO 03/29184 and WO 03/29205, e.g. compounds of formula IX

Ri f xf R3f NHZ
I CHZOR4f IX
R 2f (CH2)nf CH2OR5f wherein Xf is 0, S, SO or SO2 R,f is halogen, trihalomethyl, OH, C,-7alkyl, CI-4alkoxy, trifluoromethoxy, phenoxy, cyclohexylmethyloxy, pyridylmethoxy, cinnamyloxy, naphthylmethoxy, phenoxymethyl, CH2-OH, CH2-CH2-OH, C1_4alkylthio, CI_4alkylsulfinyl, C,-4alkylsulfonyl, benzylthio, acetyl, nitro or cyano, or phenyl, phenylC1-4aikyl or phenyl-C1_4alkoxy each phenyl group thereof being optionally substituted by halogen, CF3, CI-4alkyl or C,_aalkoxy;
R2f is H, halogen, trihalomethyl, C,-4alkoxy, C,-,alkyl, phenethyl or benzyloxy;
R3f H, halogen, CF3, OH, CI_7alkyl, C1_4alkoxy, benzyloxy or Cl_4alkoxymethyl;
each of R4fand RSf, independently is H or a residue of formula - P < ORsr OR9f O

wherein each of R8f and R9f, independently, is H or CI-4alkyl optionally substituted by halogen; and nf is an integer from 1 to 4;
e.g. 2-amino-2-[4-(3-benzyloxyphenoxy)-2-chlorophenyl]ethyl-l,3-propane-diol, 2-amino-2-[4-(benzyloxyphenylthio)-2- chlorophenyl]ethyl-1,3-propane-diol, 2-amino-2-[4-(3-benzyloxyphenoxy)-2-chlorophenyl]propyl-l,3-propane-diol or 2-amino-2-[4-(benzyloxyphenylthio)-2- chlorophenyl]propyl-1,3-propane-diol, or a pharmacological salt, solvate or hydrate thereof;

-Compounds as disclosed in WO03/062252A1, e.g. a compound of formula X
R3s /(Ras)o-a N Ar-( 9 A
CH '')"s I (CHZ)ms Ris X
wherein Ar is phenyl or naphthyl; each of mg and ng independently is 0 or 1; A is selected from COOH, PO3H2i PO2H, SO3H, PO(Cj_3alkyi)OH and 1 H-tetrazol-5-yl; each of R,9 and R29 independently is H, halogen, OH, COOH or CI-4alkyl optionally substituted by halogen; R39 is H or C1-4alkyl optionally substituted by halogen or OH; each R4g independently is halogen, or optionally halogen substituted C1_4alkyl or C1_3alkoxy; and each of Rg and M
has one of the significances as indicated for B and C, respectively, in WO03/062252A1;
or a pharmacologically acceptable salt, solvate or hydrate thereof;

-Compounds as disclosed in WO 03/062248A2, e.g. a compound of formula XI

R3h R1h (Rah)a-a A [MALRM XI
n R 2h -wherein Ar is phenyl or naphthyl; n is 2,3 or 4; A is COOH, 1H-tetrazol-5-yl, P03H2, P02H2, SO3H or PO(R5h)OH wherein R5h is selected from C1_4alkyl, hydroxyC,-,alkyl, phenyl, -CO-CI_ 3alkoxy and -CH(OH)-phenyl wherein said phenyl or phenyl moiety is optionally substituted;
each of Rlh and R2h independently is H, halogen, OH, COOH, or optionally halogeno substituted C1_6alkyl or phenyl; R3h is H or CI_4alkyl optionally substituted by halogen and/
OH; each R4h independently is halogeno, OH, COOH, C1_4alkyl, S(O)0,1 or2C1_3alkyl, C,_ 3alkoxy, C3_6cycloalkoxy, aryl or aralkoxy, wherein the alkyl portions may optionally be substituted by 1-3 halogens; and each of Rh and M has one of the significances as indicated for B and C, respectively, in WO03/062248A2;

- Compounds as disclosed in WO 04/026817A, e.g. compounds of formula XII
Rij Xj R3j NHR .
I I RSJ
A~_ Xii R2j (CHa)nj R5j R'ij wherein Rlj is halogen, trihalomethyl, C1_4alkyl, CI-,alkoxy, C1_4alkylthio, Cl_4alkylsulifinyl, C14alkyl-sulfonyl, aralkyl, optionally substituted phenoxy or aralkyloxy, R2j is H, halogen, trihalo-methyl, C14alkyl, C14alkoxy, aralkyl or aralkyloxy, R3j is H, halogen, CF3, C,_4alkyl, C14alkoxy, C1_4alkylthio or benzyloxy, R4j is H, C14alkyl, phenyl, optionally substituted benzyl or benzoyl, or lower =aliphatic C,_5acyl, R5j is H, monohalomethyl, C,_4alkyl, C1_4alkoxymethyl, C1_4alkyl-thiomethyl, hydroxyethyl, hydroxypropyl, phenyl, aralkyl, C2_4alkenyl or -alkynyl, each of R6j and R7j, independently, is H or C1_4alkyl, or R7j being also a residue of formula - P < OR8j O

wherein each of Rsj and R9j, independently, is H or C14alkyl optionally substituted by halogen Xi is 0, S, SO or SO2 and nj is an integer of 1 to 4, e.g. 2-amino-4-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]-2-methylbutane-l-ol or.2-amino-4-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]-2-ethylbutane-1-ol;
- Compounds as disclosed in WO 04/103306A, WO 05/000833, WO 05/103309 or WO
05/113330, e.g. compounds of formula Xllla or Xlllb 3k ~3k R2k Ak zk/\X\/Yk~R2k Ak ak Yk~ -~
W k N..f//
z R1 k NH
O-W__ Rlk XIIIa XIIIb wherein Ak is COOR5ki OPO(OR502, PO(OR5k)2, SO2OR5k, POR5kOR5k or 1 H-tetrazol-5-yl, R5k being H or C1_6alkyl;
Wk is a bond, C1_3alkylene or C2_3alkenylene;
Yk is C6_10aryl or C3_9heteroaryl, optionally substituted by I to 3 radicals selected from halogene, OH, NO2, C1_6alkyl, C1_6alkoxy; halo-substituted CI_6alkyl and halo-substituted Cl_6alkoxy;
Zk is a heterocyclic group as indicated in WO 04/103306A, e.g. azetidine;
Rlk is C6_10aryl or C3_9heteroaryl, optionally substituted by C1_6alkyl, C6_loaryl, C6_loarylCl_4alkyl, C3_9heteroaryl, C3_9heteroarylCl-4alkyl, C3_acycloalkyl, C3_$cycloalkylCl_4alkyl, C3_$heterocycloalkyl or C3_8heterocycloalkylC1_4alkyl; wherein any aryl, heteroaryl, cycloalkyl or heterocycloalkyl of R,k may be substituted by I to 5 groups selected from halogen, Cl_ 6alkyl, C,_salkoxy and halo substituted-C1_6alkyl or -C1_6alkoxy;
R2k is H, C1_6alkyl, halo substituted C1_6alkyl, C2_6alkenyl or C2_6alkynyl:
and each of R3k or RU, independently, is H, halogen, OH, C1_6alkyl, C1_6alkoxy or halo substituted C1_6alkyl or C1_6alkoxy;
and the N=oxide derivatives thereof or prodrugs thereof, or a pharmacologically acceptable salt, solvate or hydrate thereof.

According to a further embodiment of the invention, a S1 P receptor agonist or modulator for use in the invention may also be a selective S1 P1 receptor, e.g. a compound which possesses a selectivity for the S1 P1 receptor over the S1 P3 receptor of at least 20 fold, e.g.
100, 500, 1000 or 2000 fold, as measured by the ratio of EC50 for the S1 P1 receptor to the EC50 for the S1 P3 receptor as evaluated in a 35S-GTPyS binding assay, said compound having an EC50 for binding to the S1 P1 receptor of 100 nM or less as evaluated by the 35S-GTPyS binding assay. Representative S1 P1 receptor agonists or modulators are e.g. the compounds listed in WO 03/061567, the contents of which being incorporated herein by reference, for instance a compound of formula XIV or XV

OH
OH
cF S C ~ ~ ~ I \ H F-OH
oH O
CH3-(CHx)8 xiv or. xv When the compounds of formulae I to XV have one or more asymmetric centers in the molecule, the present invention is to be understood as embracing the various optical isomers, as well as racemates, diastereoisomers and mixtures thereof are embraced.
Compounds of formula III or lVb, when the carbon atom bearing the amino group is asymmetric, have preferably the R-configuration at this carbon atom.

The compounds of formulae I to XV may exist in free or salt form. Examples of pharmaceutically acceptable salts of the compounds of the formulae I to XIII
include salts with inorganic acids, such as hydrochloride, hydrobromide and sulfate, salts with organic acids, such as acetate, fumarate, maleate, benzoate, citrate, malate, methanesulfonate and benzenesulfonate salts, or, when appropriate, salts with metals such as sodium, potassium, calcium and aluminium, salts with amines, such as triethylamine and salts with dibasic amino acids, such as lysine. The compounds and salts of the combination of the present invention encompass hydrate and solvate forms.

Acyl as indicated above may be a residue R, CO- wherein R,, is C1.6alkyl, C3.6cycloalkyl, phenyl or phenyl-C1.4alkyl. Unless otherwise stated, alkyl, alkoxy, alkenyl or alkynyl may be straight or branched.

When in the compounds of formula I the carbon chain as R, is substituted, it is preferably substituted by halogen, nitro, amino, hydroxy or carboxy. When the carbon chain is interrupted by an optionally substituted phenylene, the carbon chain is preferably unsubstituted. When the phenylene moiety is substituted, it is preferably substituted by halogen, nitro, amino, methoxy, hydroxy or carboxy.

Preferred compounds of formula I are those wherein R, is C13.20alkyl, optionally substituted by nitro, halogen, amino, hydroxy or carboxy, and, more preferably those wherein R, is phenylalkyl substituted by C6.14-alkyl chain optionally substituted by halogen and the alkyl moiety is a C1.6alkyl optionally substituted by hydroxy. More preferably, R, is phenyl-C,_6alkyl substituted on the phenyl by a straight or branched, preferably straight, C6_14alkyl chain. The C6_14alkyl chain may be in ortho, meta or para, preferably in para.

Preferably each of R2 to R5 is H.

A preferred compound of formula I is 2-amino-2-tetradecyl-1,3-propanediol. A
particularly preferred S1 P receptor modulator of formula I is FTY720, i.e. 2-amino-2-[2-(4-octylphenyl) ethyl]propane-1,3-diol in free form or in a pharmaceutically acceptable salt form (referred to hereinafter as Compound A), e.g. the hydrochloride, as shown:

HO OH
HzN HCI

A preferred compound of formula II is the one wherein each of R'2 to R'5 is H
and m is 4, i.e.
2-amino-2-{2-[4-(1-oxo-5-phenylpentyl)phenyl]ethyl}propane-l,3-diol, in free form or in pharmaceutically acceptable salt form (referred to hereinafter as Compound B), e.g. the hydrochloride.

A preferred compound of formula III is the one wherein W is CH3, each of R", to R"3 is H, Z2 is ethylene, X is heptyloxy and Y is H, i.e. 2-amino-4-(4-heptyloxyphenyl)-2-methyl-butanol, in free form or in pharmaceutically acceptable salt form (referred to hereinafter as Compound C), e.g. the hydrochloride. The R-enantiomer is particularly preferred.

A preferred compound of formula IVa is the FTY720-phosphate (R2a is H, R3a is OH; Xa is 0, Ria and Rib are OH). A preferred compound of formula IVb is the Compound C-phosphate (R2a is H, R3b is OH, Xa is 0, Ria and Rib are OH, Ya is 0 and R4a is heptyl).

A preferred compound of formula VIII is (2R)-2-amino-4-[3-(4-cyclohexyloxybutyl)-benzo[b]thien-6-yl]-2-methylbutan-l-ol.

A preferred compound of formula IX is a compound wherein Xf is S or 0, R,f is benzyloxy, Rzf, R4f and R5f are each H, R3f is Cl and nf is 2.

A preferred compound of formula XII is a compound wherein Xj is S or 0, Ri; is benzyloxy, R2j, R4j, R6j and R7j are each H, R3j is Cl, R5j is hydroxyethyl or hydroxypropyl and nj is 2.
A preferred compound of formula XIIla is e.g. 1-{4-[1-(4-cyclohexyl-3-trifluoromethyl-benzyloxyimino)-ethyl]-2-ethyl-benzyl}-azetidine-3-carboxylic acid, or a prodrug thereof.

The immunosuppressive drug or drugs to be combined with the S1 P receptor modulator or agonist are e.g. a calcineurin inhibitor; a mTOR inhibitor, mycophenolic acid, a salt or a prodrug thereof e.g. mycophenolate sodium or mycophenolate mofetil, or a steroid, e.g.
prednisone, methylprednisolone, dexamethasone, triamcinalone acetinide and the like.

As used herein the term "calcineurin inhibitors" includes e.g. a cyclosporin, e.g. cyclosporin A or ISA tx 247 or FK506 (Tacrolimus).

As used herein the term "mTOR inhibitor" includes rapamycin or a derivative thereof which are thought to have the same mechanism of action (e. g., inhibition of mTOR
activity) and have immunosuppressive properties. Suitable derivatives of rapamycin include e.g.
compounds of formula A

H,CO 9 38 37 36 =

7 2 1 ~ Xõ 28 OH

ON'9 O 26 O~ Riea 24 11 = 18 20 22 13 15 19 21 ?
wherein Rlaa is CH3 or C3_6alkynyl, R2aa is H or -CH2-CH2-OH, 3-hydroxy-2-(hydroxymethyl)-2-methyl-propanoyl or tetrazolyl, and Xea-is--=0; -(H;H)-or (H,OH) .
provided that R2aa is other than H when Xaa is =0 and Rlaa is CH3.
or a prodrug thereof when R2aa is -CH2-CH2-OH, e.g. a physiologically hydrolysable ether thereof, e.g. a compound wherein R2aa is -CH2-CH2-O-Alk, Alk being a Cl_9alkyl optionally interrupted in the chain by 1 or 2 oxygen atoms.

Compounds of formula A are disclosed e.g. in WO 94/09010, WO 95/16691, WO
96/41807, USP 5,362,718 or WO 99/15530 which are incorporated herein by reference. They may be prepared as disclosed or by analogy to the procedures described in these references.
Preferred rapamycin derivatives are 32-deoxorapamycin, 16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32(S)-dihydro-rapamycin, 16-pent-2-ynyloxy-32(S)-dihydro-40-0-(2-hydroxyethyl)-rapamycin and, more preferably, 40-0-(2-hydroxyethyl) rapamycin. Further examples of rapamycin derivatives include e.g. CC1779 or 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin or a pharmaceutically acceptable salt thereof, as disclosed in USP 5,362,718, ABT578 or 40-(tetrazolyl)-rapamycin, particularly 40-epi-(tetrazolyl)-rapamycin, e.g. as disclosed in WO 99/15530.
Rapamycin derivatives may also include the so-called rapalogs, e.g. as disclosed in WO
98/02441, WO01/14387 and WO 03/64383, e.g. AP23573, AP23464, AP23675 or AP23841. Further examples of a rapamycin derivative are those disclosed under the name TAFA-93, biolimus-7 or biolimus-9.

According to the invention, the antilymphocyte antibody may be administered at various time points during the immunosuppressive treatment of a transplant recipient, e.g.
weeks, months or even years after transplantation, and/or prior to transplantation and/or immediately after transplantation, to induce alteration of the immune response to enhance graft acceptance.
Antilymphocyte antibodies include e.g. polyclonal antibodies such as antilymphocyte globulin, anti-thymocyte globulins ("ATGs"), e.g. whether from rabbits or horses, ATGAM
and Thymoglobulin ; monoclonal antibody preparations such as antibodies, e.g.
chimerized, humanized or human, to leucocyte receptors, e.g. CD2, CD3, CD4, CD7, CD25, CD27, CD28, CD40, CD45, CD80, CD86, ICOS, OXA40, or to their ligands, e.g. CD154, for example OKT3, muromonab-CD3, basiliximab (Simulect ; Novartis AG, CH) and daclizumab (Zenapax ; Roche AG, CH); or a recombinant binding molecule having at least a portion of the extracellular domain of CTLA4 or a mutant thereof, e.g. an at least extracellular portion of CTLA4 or a mutant thereof joined to a non-CTLA4 protein sequence, e.g. CTLA4-Ig (for ex. designated ATCC68629) or a mutant thereof, e.g. LEA29Y
(belatacept). Particularly preferred monoclonal antibodies are anti-CD25 either chimeric (for example, 'as described in detail in EP 449769 the contents thereof being included herein by reference) or humanized (for example, as described in detail in WO 90/07861, the contents thereof being incorporated herein by reference).

In a series of further specific or alternative embodiments, the present invention also provides:

1.1. A method for inhibiting allograft rejection in a recipient, said method comprising administering to said recipient, e.g. simultaneously or sequentially, a therapeutically effective amount of a S1 P receptor modulator in combination with one or more immunosuppressive drug(s) and an antilymphocyte antibody.

Preferably the method is used to prevent or treat organ graft rejection in a solid organ graft recipient. Preferred S1 P receptor modulator is Compound A, B or C, (2R)-amino-4-[3-(4-cyclohexyloxybutyl)-benzo[b]thien-6-yl]-2-methylbutan-1-ol, or a-compound of formula IX wherein Xf is S or 0, Rlf is benzyloxy, R2f, R4f and R5f, are each H, R3f is Cl and nf is 2, or a compound of formula XIIia.

Preferred immunosuppressive drugs for use in a method according to the invention are e.g.

- a calcineurin inhibitor, for example cyclosporin A or FK506, optionally in combination with a steroid, e.g. a corticosteroid, for example prednisone; or - a mTOR inhibitor, for example everolimus or sirolimus, optionally in combination with a steroid, e.g. a corticosteroid, for example prednisone; or - a mTOR inhibitor, for example everolimus or sirolimus, optionally in combination with a calcineurin inhibitor, for example cyclosporin A or FK506, and a steroid, e.g. a corticosteroid, for example prednisone; or - mycophenolic acid, or a salt or a prodrug thereof, optionally in combination with a calcineurin inhibitor, for example cyclosporin A or FK506, and a steroid, e.g.
a corticosteroid, for example prednisone; or - mycophenolic acid, or a salt or a prodrug thereof, optionally in combination with a steroid, e.g. a corticosteroid, for example prednisone.

1.2 A method for inhibiting allograft rejection in a recipient, said method comprising administering to said recipient simultaneously or sequentially a therapeutically -- --, ~._ - - effective amount of a SIP receptor modulator in combination with one or more immunosuppressive drug(s) and an antilymphocyte antibody, wherein the antilymphocyte antibody is administered prior to transplantation and/or shortly after transplantation.

The antilymphocyte antibodies may be administered for the so-called induction treatment, i.e. as short term treatment for single or multiple administration in the very early phase following transplantation, e.g. shortly before the transplantation and up to 3 months after transplantation. Preferred S1 P receptor agonists or modulators and preferred immunosuppressive drugs are e.g. as indicated above.

1.3. A method for inhibiting allograft rejection in a recipient, said method comprising administering to said recipient simultaneously or sequentially a therapeutically effective amount of a S1 P receptor modulator in combination with one or more immunosuppressive drug(s) combined with an antilymphocyte antibody induction comprising administration at least prior to transplantation of an anti-CD25 compound or a recombinant binding molecule having at least a portion of the extracellular domain of CTLA4 or a mutant thereof.

2. The use of a S 1 P receptor modulator in combination with one or more immunosuppressive drug(s) and an antilymphocyte antibody, in the manufacture of a medication for use in any method as defined in 1.1 to 1.3 above, e.g.
inhibiting allograft rejection in a recipient, whereby said medication is administered simultaneously or sequentially.

3. A combination, e.g. a kit for use in any method as defined in 1.1 to 1.3 above, e.g. in the treatment of an allograft transplant recipient, comprising a S1 P receptor modulator, one or more immunosuppressive drug(s) and an antilymphocyte antibody.

The method of the invention is indicated e.g. in solid organ transplant, e.g.
kidney, heart, lung or liver transplants, preferably kidney transplants.

The effective dosage of each of the combination partners employed in the method of the invention may vary depending on the particular compound or pharmaceutical composition employed, the mode of administration, the severity of the condition being treated. A
physician or clinician of ordinary skill can readily determine and prescribe the effective amount of the single active ingredients required.

'Daily dosage of the S1 P receptor modulator may vary between 0.5 and 15 mg.
Preferred dosages are of from 1 to 15 mg/day, more preferably 2 to 5 mg/day, p.o.
including the day prior to transplantation. More preferably the S1 P receptor modulator is administered orally at a dose of 5 mg the day prior to the transplantation and then at a dose of from 2.5 mg/day.
The dose of Cys A may be 0.5-10 mg/kg/day p.o., depending upon time after transplantation, and with or without blood level monitoring.

The dose of FK506 may be 0.05 to 0.2 mg/kg/day p.o.

When the antibody is basiliximab or daclizumab, it may be administered at a single dose of about 2mg/kg or at a dose of 4x about 1 mg/kg, e.g. 2mg/kg pre-transplantation followed by 4 additional doses of 1 mg/kg at 2 weekly intervals. Thymoglobulin or lymphoglobulin may be administered at a dose of 1-3mg/kg. Administration of the antilymphocyte antibody may be e.g. on a weekly or monthly basis, for example every week, every two, three, four, five, six, seven or eight weeks, regularly or irregularly, as required. LEA29Y may be administered at periodic intervals in varying doses, e.g. at pre-transplantation with a dose of 10mg/kg, at day and every 2 weeks for 3 months and then monthly thereafter with a dose reduced to e.g. 5 mg/kg at month 7.

The mTor inhibitor may be administered at a daily dose of about 0.5 to 30mg, optionally in divided doses. Mycophenolic acid, salt or prodrug thereof may be administered at a daily dose of about 150mg to 3g, optionally in divided form.

Utility of the combination of a S1 P receptor modulator, an immunosuppressive drug and an antibody for induction in treating diseases and conditions as hereinabove specified may be demonstrated in standard animal or clinical tests, e.g. in accordance with the methods described hereinafter.

Study:
The patients are randomized to one of two treatment groups:

Group 1: FTY720 5 mg*, then 2.5 mg QD + cyclosporine A, 8-10 mg/kg/day adjusted to achieve target blood levels + corticosteroids**

Group 2: FTY720 5 mg*, then 5 mg QD + cyclosporine A, 3-4 mg/kg/day adjusted to achieve target blood levels + corticosteroids**

*The first dose of FTY720 is given 2 to 12 hours prior to renal allograft revascularization.
Day 0 is defined as the day of administration of the first dose of study medication.

**In addition to one of the above treatment regimens, all patients receive antibody induction (anti-CD25) prior to transplantation, e.g. at a dose of 2mg/kg. Patients may further receive 4 additional doses of 1 mg/kg at 2 weekly intervals of anti-CD25.

Cys A target ranges for Group 1 Study time Target Cs A Group 1(ng/mL) Month 1 1000-1500 Month 2 800-1200 Months 3-12 600-1000 Cys A target ranges for Group 2 Study time Target Cs A Group 2 (ng/mL) Month 1 800-1200 Months 2 600-800 Months 3-12 400-600 Maintenance treatment with FTY720 commences after graft revascularization which occurs either on Day 0 or Day 1, between 12 and 24 hours after the first dose.

Patients randomized to Group 1 start Cyclosporine A 8-10 mg/kg/day in two divided doses adjusted to achieve the target blood levels and those randomized to Group 2 start Cyclosporine A 3-4 mg/kg/day in two divided doses adjusted to achieve the target blood levels.

During the 12 month treatment period, patient visits occur on Days 0, 1, 3, 5, 7 (or day of discharge from the hospital if sooner than 7 days), 14 and 28, and Months 2, 3, 6, 9 and 12.
An interim safety analysis is performed when all patients complete 3 months on study. The final analysis of safety and efficacy is performed when all patients have completed 12 months on study.

Key safety assessments:
Adverse events/serious adverse events Infections/serious infections Discontinuation of study medication due to adverse event (including infection) or laboratory abnormality Other assessments:
Incidence of treated biopsy proven acute rejection Incidence of graft loss Incidence of death Incidence of malignancy HCV viral load BK polyoma viral load The dosage regimen of the study has a beneficial effect compared to standard immunosuppressive regimens. Depending on the regimen, monitoring of drug levels becomes less mandatory and fixed dose treatment may become possible.

The clinical trial above may be repeated using a different daily dose of cyclosporine A, e.g.
3-6 mg/kg administered in 2 divided doses, e.g. in Group 2.
The clinical trial above may be repeated using a different S1 P receptor agonist or modulator, e.g. a compound of formula IX or a compound of formula Xllla.
The clinical trial above may be repeated using everolimus instead of cyclosporine A.
The clinical trial above may be repeated using LEA29Y instead of the anti-CD25.

Claims (11)

1. The use of a S1P receptor modulator in combination with one or more immunosuppressive drug(s) and an antilymphocyte antibody, in the manufacture of a medication for inhibiting rejection of a solid organ allograft in a recipient, whereby said medication is administered simultaneously or sequentially.

2. The use according to claim 1 wherein the antilymphocyte antibody is administered prior to transplantation and/or shortly after transplantation.

3. The use according to claim 2 wherein the antilymphocyte antibody is administered prior to transplantation, the antilymphocyte antibody being selected from an anti-CD25 compound and a recombinant binding molecule having at least a portion of the extracellular domain of CTLA4 or a mutant thereof.

4. The use according to claim 1 wherein the solid organ allograft is a kidney.

5. A method for inhibiting rejection of a solid organ allograft in a recipient, said method comprising administering to said recipient, simultaneously or sequentially, a therapeutically effective amount of a S1P receptor modulator in combination with one or more immunosuppressive drug(s) and an antilymphocyte antibody.

6. A method for inhibiting rejection of a solid organ allograft in a recipient, said method comprising administering to said recipient simultaneously or sequentially a therapeutically effective amount of a S1P receptor modulator in combination with one or more immunosuppressive drug(s) and an antilymphocyte antibody, wherein the antilymphocyte antibody is administered prior to transplantation and/or shortly after transplantation.

7. A method according to claim 6 wherein the antilymphocyte antibody is administered prior to transplantation, the antilymphocyte antibody being selected from an anti-CD25 compound and a recombinant binding molecule having at least a portion of the extracellular domain of CTLA4 or a mutant thereof.

8. A combination for inhibiting rejection of a solid organ allograft in a recipient comprising a S1P receptor modulator, one or more immunosuppressive drug(s) and an antilymphocyte antibody.

9. The use according to claim 1, a method according to claim 5 or 6 or a composition according to claim 8, wherein the S1P receptor modulator is selected from a compound of formulae I to XV as hereinbefore defined, in free form or in a pharmaceutically acceptable salt form.

10. The use according to claim 1, a method according to claim 5 or 6 or a composition according to claim 8, whereby the immunosuppressive drug is selected from a calcineurin inhibitor, a mTOR inhibitor, mycophenolic acid, a salt or a prodrug thereof and a steroid.

11. The use according to claim 1, a method according to claim 5 or 6 or a composition according to claim 8, whereby the antilymphocyte antibody is an anti-CD25 antibody, an antilymphocyte globulin or an anti-thymocyte globulin or a recombinant binding molecule having at least a portion of the extracellular domain of CTLA4 or a mutant thereof.